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Integrated Technologies for the
Characterization of
Phosphodiesterase (PDE) Inhibitors
11 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer
Phosphodiesterase (PDE) Inhibitors
Edmond Massuda, Laurel Provencher, Abbie Esterman,
Benjamin Lineberry, Lisa Fleet, Chris Spence, Dhanrajan
Tiruchinapalli, Scott Perschke, Hao Chen, and Seth Cohen
Caliper Discovery Alliances and Services (CDAS)
PKI Global Health Summit - June 8, 2012
PerkinElmer Accelerates Drug Discovery
Molecular Screening
Mechanism of Action
Compounds Libraries
Drug Safety Assessment
Selectivity PanelsAssay Development
Pathway Analysis
22
Mechanism of Action
In Vivo Imaging Pre-Efficacy StudiesPK-ADME
Cellular Functional Assay
Drug Combinations
Marker Identification/Detection
Ex-vivo tissue analysis
LabChip™ Mobility-Shift Assay Target Classes
� Epigenetic Targets
– HATs– HDACs– Methyl Transferases– Demethylases
� Kinases
� Phosphodiesterases
� Phosphatases
� Proteases
� Lipid Kinases
� DNA/RNA Binding Proteins
Kinetic or endpoint assays for target classes including:
33
� DNA/RNA Binding Proteins
Phosphodiesterases are enzymes that play a major role in cell
signaling and function, by regulating levels of the second
messengers cAMP and cGMP
Introduction
Dual
SpecificitycAMP
44
PDE4B(catalytic domain)
PDE4
PDE7
PDE8
PDE1
PDE2
PDE3
PDE10
PDE11
PDE5
PDE6
PDE9
SpecificitycAMP
Specific
cGMP
Specificlocation, duration, amplitude
Success of therapeutic inhibitors has validated PDEs as important drug targets
� Viagra/Cialis/Levitra (PDE5)
� Milrinone – heart failure (PDE3)
� Cilostazol – claudication (PDE3)
� Dipyridamole – anti platelet aggregation, stroke
Studies in clinical trials:
Clinical PDE Inhibitors
55
Studies in clinical trials:
� TPI1100 – inflammation
– PDE4/7 inhibitor (Topigen)
� IC224 series – ADHD, Parkinson’s
– PDE1 inhibitors (ICOS/Lilly)
� NDxxxx series – depression, chronic inflammation, atherosclerosis
– PDE4 inhibitors (Via Pharma, Celgene, and Nycomed)
� PF-2545920 – schizophrenia
– PDE10 inhibitors (Pfizer)
Regulate second messengers
High PDE activity in many tissues
Many isoforms connected to different physiological functions
� 21 PDE gene products
PDE’s potential as drug targets
66
� 21 PDE gene products
� Isoform selective inhibitors
PDE’s relatively unique in substrate binding
Caliper Life Sciences LabChip® Platform
Substrate
Key benefits:
� No antibodies or radioactivity
� Stopped rxn or real-time kinetic measurements
77
Product� Stopped rxn or real-time kinetic measurements
� Superior data quality:
- Direct measurement of substrates & products
- Ratiometric method
- High signal-to-noise ratios
- Fewer false positive and negative
� Screening with cAMP/cGMP around PDE Km
� HTS assay format (384 wells)
� Activation assays also available
Assay Principle – Substrate/Products structures
� Hydrolization of Labeled cAMP and cGMP creates charge difference with products
� Allows for independent detection of substrates and products
� Enzyme percent conversion is defined as a measure of ration P/(P+S)
cAMP
S
S
88
MW 942.9iFL-cGMP
cGMP
SS
P
MW 926.9iFL-cAMP
P
Reaction volume of 25 µµµµL in a 384 well plate
PDE enzymes available from BPS Bioscience, Inc
iFL-cAMP or iFL-cGMP substrate (Caliper)
Substrate concentrations near or below substrate Km
PDE LC Assay: Materials and Methods
99
Substrate concentrations near or below substrate Km
Reaction buffer: 100 mM Hepes pH 7.5, 5 mM MgCl2, 0.002% Brij-35
One hour incubation time at room temperature
Caliper’s Reviewer software measures % conversion
1010 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer
PDE10A cAMP Km
0.1
0.2
0.3
V (
pm
ol/
min
)
PDE1A cGMP Km
0.50
0.75
1.00
1.25
V (
pm
ol/
min
)PDE Substrate Km’s
1111
0 1 2 3 4 50.0
0.1
VMAX
KM
A
0.2503
0.3602
[cAMP] (uM)V
(p
mo
l/m
in)
0 5 10 15 200.00
0.25
VMAX
KM
A
1.477
6.230
[cGMP] (uM)
V (
pm
ol/
min
)
Substrate Km determination for PDE1A with cGMP, and PDE10A with cAMP. These are representative of the 20 PDE substrate Km’s tested.
Determination of inhibitor IC50s for various PDEs.
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
% S
pecific
Activity
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
log [drug] (M)
% S
pecific
Activity
PDE1A
cGMP
PDE10A
cAMP
1212
Trequinsin
Reference Compound IC50 (nM)
1,800
22,000
26,000
>100,000
>100,000
8-methoxy-IBMX
Zaprinast
Pentoxifylline
Rolipram
log [drug] (M)
Trequinsin
Rolipram
Reference Compound IC50 (nM)
70
2,700
3,500
19,000
96,000
>100,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Reference Compound IC50 (nM)
1,300
10,000
25,000
25,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Reference Compound IC50 (nM)
200
770
1,900
5,500
44,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE2A
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Reference Compound IC50 (nM)
590
3,400
6,300
41,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
log [drug] (M)
% S
pec
ific
Ac
tivit
y
PDE3A
-13 -12 -11 -10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Rolipram
Reference Compound IC50 (nM)
0.16
4,800
93,000
>100,000
>100,000
>100,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Rolipram
Reference Compound IC50 (nM)
240
490
4,900
>100,000
>100,000
>100,0008-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Rolipram
Reference Compound IC50 (nM)
374
1,050
3,960
42,000
53,000
91,0008-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE4D2
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Rolipram
Reference Compound IC50 (nM)
150
630
2,800
30,000
30,000
55,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE7A1
-9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Rolipram
Reference Compound IC50 (nM)
5,500
8,400
>100,000
>100,000
>100,000
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE9
-9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Reference Compound IC50 (nM)
15,000
17,000
>100,000
>100,000
8-methoxy-IBMX
Zaprinast
Rolipram
Dipyridamole
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE4D3
PDE11A4
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Rolipram
Reference Compound IC50 (nM)
57
136
1,400
5,400
50,000
50,000
8-methoxy-IBMX
Dipyridamole
Zaprinast
Pentoxifylline
log [drug] (M)
% S
pecif
ic A
cti
vit
y
-10 -9 -8 -7 -6 -5 -4
-10
0
10
20
30
40
50
60
70
80
90
100
110
Trequinsin
Reference Compound IC50 (nM)
660
>100,000
>100,000
>100,000
8-methoxy-IBMX
BRL-50481
Rolipram
log [drug] (M)
% S
pe
cif
ic A
cti
vit
y
PDE4B1PDE4A1A
PDE5A1 PDE8A1PDE Panel:now 20 assays
Compound Specificity
•Compound specificity critical to drug development
•Side effects
1313
0.16 nM (PDE3) 100 uM (PDE7)
DMSO Tolerance
40
60
80
100PDE 3A
PDE 5A
PDE 10A
% A
cti
vit
y
Assay DMSO Sensitivity
1414
0.0
0.7
1.4 2.1 2.8 3.4
4.1
5.4
6.7
7.9
0
20
40
% DMSO
% A
cti
vit
y
Concentrations of DMSO were analyzed
for each assay. These are representative of the 20 PDE DMSO tolerances tested.
•Limit PDE assays to
1% DMSO if possible
Z Prime
PDE Z’
PDE1A 0.63
PDE2A 0.69
PDE3A 0.74
PDE4A1A 0.74
PDE4B1 0.80
PDE4B Z Prime
Z'=0.80
30
40
50
% C
on
ve
rsio
n%
Convers
ion
PDE4B1 Z Prime
1515
Z prime determination for PDE4B1.
PDE4B1 0.80
PDE4D2 0.82
PDE4D3 0.86
PDE5A1 0.74
PDE7A1 0.63
PDE8A1 0.81
PDE9A2 0.62
PDE10A1 0.70
PDE11A4 0.75
Average 0.73
0
10
20
0 50 100 150 200
Well Number
% C
on
ve
rsio
n%
Convers
ion
Collaborating with customers to improve the health and safety of people and their environment
1616 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer
In-Depth PDEi Research
Lead Optimization: Compound Evaluation Flowchart
IC50 Determination
Tight-Binding?
NOYES
Linear Progress Curve
Reversible
Covalent Inhibition
Biochemical analyses typically performed during the characterization of lead compounds for
drug discovery
1717
YESYES
YES
NO
NO
NO
Linear Progress Curve Covalent Inhibition
Poor
CandidateFurther Characterization
Mechanism BasedClassical Steady-State
Anaysis
Time-Dependent
Inhibition Analysis
Adapted from Copeland, R.A. (2005)
Trequinsin ReversibilityPDE3A
10
20
30
40
50 0X
10X
30X
100X
[Trequinsin], asmultiple of IC50
% C
on
ve
rsio
n
Trequinsin Linearity
40
50
1x
0.5x
0
[Trequinsin] asmultiple of IC50
% C
on
ve
rsio
n
Linear inhibition over timeof PDE3A by Trequinsin
0 25 50 75 100 1250
15
30
45
60
.
Time, Min
•time dependent example
•no time dependence
PDE3A Mechanism of Action Study
1818
0 10 20 30 40 50 600
Time, minutes
Determination of reversibility
for Trequinsin for PDE3AEnzyme is incubated with inhibitor
concentration indicated above and 100X
enzyme. Assay is then diluted to 1X
enzyme with substrate. For reversible
inhibitors, conversion rate after
10X→0.1X inhibitor dilution is restored.
Determination of reaction progress linearity with TrequinsinReaction linearity with inhibitor is a prerequisite for determination of the Ki and mode of inhibition.
0 10 20 30 40 50 60 70 80 90 1000
10
20
30
4x
2x
1x
Time, minutes
% C
on
ve
rsio
n
PDE3A Mechanism of Action Study
PDE3A Curve FitTrequinsin
10
15
20
40
0
80
Trequinsin (nM)
V (
pm
ol/m
in)
1919
0 1 2 3 4 5 6 70
5
10
VMAX
KM
0
21.04
0.7232
40
17.11
0.8597
80
19.30
1.307
160
15.91
2.361
320
11.78
4.186
480
4.385
2.187
160
320
480
[cAMP] (uM)
V (
pm
ol/m
in)
Michaelis-Menton Plot of Trequinsin for PDE3A
Inhibitor and substrate concentrations are varied around the IC50 and Km, respectively.
PDE3A Mechanism of Action Study
Trequinsin Ki at PDE3A
2
3
4
5
Slope 0.011Y-int = 0.53K
m,
uM
0.6
0.8
1.0
1.2
1.4Lineweaver-Burk
1/V
o
[I] = 0.00
[I] =40.00
[I] =80.00
[I] =160.00
[I] =320.00
[I] =480.00
0
40
80
160
320
480
Lineweaver-Burk Plot
Trequinsin with PDE3A Trequinsin (nM)
2020
0 70 140 210 280 3500
1
2 Y-int = 0.53Ki = 46 pM
r2 = 0.99
[Trequinsin] (pM)
Km
, u
M
- Determination of Trequinsin Ki for PDE3A and mode of inhibition.
- Data is plotted in a Lineweaver-Burk Plot and the Ki is determined by slope of Km vs [I].
Competitive inhibition-2 -1 0 1 2 3
-0.2
0
0.2
0.4
1/[S]
Ki and kinact determination using 3D Fit Modeling
Ki 1.72E-09 (M)
k
Input:
IC50 + time
Output:
Analysis of irreversible inhibitors
2121
kinact 1.03E-02 (min-1)
Time dependent enzyme inactivation with a PDE4 inhibitor and determination of the Ki and kinact
using the Krippendorff equation, above (J Biomol Screen. 14, 2009, pp. 913-923). Ki and kinact
were estimated using XLfitTM (IDBS) and 3D Fit modeling with the Krippendorff equation.
Kinact – inactivation rate
constant at ∞ inactivation concentration
PDE Cellular Assay Characterization
2222
Schematic diagram of a PDE cellular assay using a transfected CRE-Luciferase reporter construct to measure cAMP signaling.
PDE2A cellular inhibition measured in HEK 293 cells co-transfected with a PDE2A expression vector and a CRE-Luciferase construct.Luciferase activity determined after 48 hours
PDE Immunoreactivity Assay
Low-resolution raw 4X image showing
PDE2 immunoreactivity in coronal section
of mouse brain.
2323
PDE2 immunoreactivity in coronal section of mouse brain, with discrete staining pattern in many regions. (i)
Section of the mouse brain shows prominent expression of PDE2A in the forebrain structures, including the
cortex, piriform cortex, hippocampus and Habenulo-interpeduncular. These 20X high-resolution images were
spectrally unmixed using Vectra/Nuance image-analysis system (Caliper LifeSciences/PerkinElmer, Inc.).
PDEScreen™ Research Services from CDAS
PDEScreen™ HTS� Customized screening projects to meet
clients’ expectations of throughput, process & workflow.
� 2 week turnaround
PDEScreen™ IC50� Compound inhibitor potency
A complete suite of PDE offerings
2424
� Compound inhibitor potency determined in your selection of PDE assays.
PDEScreen™ PROFILEing� Extensive panel of 20 human PDE
assays
PDEScreen™ Mechanism of Action (MOA)� Understand the manner in which your
compounds interact with enzymatic targets.
Caliper Discovery Alliances &
Services
Discovery and Profiling services
>1000 in vitro / 100 in vivo assays
The Labchip® EZ Reader II platform enables high quality and high throughput PDE
enzyme assays
Fluorescent analogs of cAMP and cGMP can be utilized as substrates for PDE
assays and are available directly from Caliper Life Sciences/PerkinElmer, Inc.
Mechanism of action of PDE inhibitors can be determined with Labchip®
technology
Summary
2525
PDE2 immunostaining in the mouse brain was acquired using the Vectra/Nuance
Imaging system (Caliper Life Sciences/PerkinElmer, Inc). PDE2 showed discrete
staining in specific regions of the brain
Multiplex tissue imaging can correlate PDE and CREB to allow pharmacodynamic
characterization of drug effects
A complete suite of contract research services for PDE drug discovery and
development is available (20 PDE’s)
For more information, please contact:
Caliper Discovery Alliances & Services:
or visit http://www.caliperls.com/products/contract-research/
2626
or visit http://www.caliperls.com/products/contract-research/